The present invention relates to an enzymatic composition and an aqueous bath for seasoning or maturing green wood, and also to a method using this enzymatic composition or this bath.
The use of wood as a material in construction, carpentry or cabinet-making presumes prior drying in order to dimensionally stabilize this wood. For cooperage, there are also organoleptic requirements.
For most industrial applications, this drying takes place in a drying facility at controlled temperature and humidity.
For other applications, in particular when the wood will subsequently be in contact with foods, the drying takes place naturally, outside, over quite long periods of time, with the aim of eliminating certain undesirable compounds. The duration of this natural drying, or ageing, can be varied depending on the final use. This drying can take up to 2 years in order to obtain good organoleptic qualities.
In the case of natural drying, the wood is colonized by a fungal flora which can transform certain compounds of the wood. These transformations of the wood cannot, however, be controlled. Naturally dried wood exhibits great variability of quality.
On the other hand, with artificial drying in a drying facility, these transformations do not occur and the wood thus dried is not suitable for food applications since this drying does not make it possible to eliminate certain undesirable molecules (such as toxic or bitter molecules).
EP 0 001 540 A relates to the use of microorganisms or of enzymes (pectinase or cellulase) with the aim of modifying the structure of wood intended for decoration, by controlling the temperature, the water content of the wood, and the O2 and CO2 content in the wood""s environment. In this method, the wood is pretreated before inoculation, post treated in order to terminate the microbiological process, and then dried.
This complex method does not use immersion of the wood in an enzymatic preparation.
DD 292 864 A relates to improving the impregnability of the sapwood of resinous wood using an enzymatic preparation (pectinase, cellulase and hemicellulase), at the same time as an aqueous mineral preparation intended to superficially protect this wood.
This method does not relate to the duramen of the wood, or to species other than conifers, or to a food application.
FR 2 421 039 A relates to the conditioning of wood in a solid medium, using microorganisms, with a view to facilitating the fragmentation and then the agglomeration of the elements of the wood.
This method does not use immersion of the wood or an enzymatic preparation, and does not relate to application in the food industry.
FR 2 705 607 A relates to the use of microorganisms having xcex1-amylase and xcex2-glucosidase activities, with a view to improving the organoleptic qualities of oak intended for cooperage. The microbiological process is carried out in the solid state using a fungal leaven. The process, which is supposed to reproduce the effects of natural drying/seasoning, specifies neither the duration of bringing the microorganisms into contact with the wood, nor the temperature, nor the microorganism content. It does not describe a method of immersion of the wood, nor an enzymatic preparation, and it applies only to species of oak.
The aim of the present invention is to provide a method for seasoning wood which allows, once it has been used, notably shortened drying of the wood. This method is suitable for food applications and conserves the dimensions of treated wood.
A subject of the invention is an enzymatic composition for drying wood, comprising mainly enzymes of the cellulase class, and also an aqueous bath for seasoning wood, comprising an endocellulase activity of between 1xc3x97106 and 100xc3x97106 ECU (preferably between 3xc3x97106 and 30xc3x97106 ECU) per m3 of wood to be treated, xylanase activity of between 0.6xc3x97106 and 60xc3x97106 BXU (preferably between 2xc3x97106 and 20xc3x97106 BXU) per m3 of wood to be treated, a xcex2-mannanase activity of between 0.4xc3x97106 and 40xc3x97106 MNU (preferably between 1xc3x97106 and 10xc3x97106 MNU) per m3 of wood to be treated and an xcex1-amylase activity of between 1xc3x97106 and 100xc3x97106 xcex1TU (preferably between 3xc3x97106 and 30xc3x97106 xcex1TU) per m3 of wood to be treated.
A subject of the invention is also a method for seasoning wood, prior to drying, comprising a step of bringing the wood into contact with an aqueous bath comprising the composition according to the invention.
Particular embodiments of the invention are described in the dependent claims.
The enzymatic composition used may comprise enzymes of the exocellular or endocellular type. The enzymes may be of bacterial or fungal origin, or of any other possible origin.
The wood to be treated, the water content of which has ideally been brought to between 25 and 45% by being rapidly passed through a drying facility, is immersed in a bath containing water or any other suitable solvent, and varying amounts of enzymes mainly of the hydrolase class (International Union of Biochemistry (IUB) class 3), chosen from enzymes of the cellulase type (also known under the name glucosidases) (IUB classification subclass 3.2). In addition, enzymes of the lipase (subclass 3.1), peroxidase (subclass 1.11.1), etc. type, and mixtures of these enzymes, may be added. Among cellulases which can be used for the present invention, mention may in particular be made of cellobiohydrolases, endoglucanases, xcex2-glucosidases, hemicellulases, xcex1-amylases, and xylanases, endocellulases, xcex2-mannanases, etc., without losing sight of the fact that the enzymes often have secondary activities besides their main activity.
Depending on whether neutral or acid cellulases are used, the pH of the bath is adjusted to between 3 and 8, preferably between 4 and 7. The temperature of the bath is kept constant at between 20 and 70xc2x0 C., preferably between 30 and 50xc2x0 C. After this treatment by immersion, which can last from 30 minutes to 2 weeks, preferably from 1 hour to 1 week, depending on the strength of the effect desired, on the mixture of enzymes used, on the volume to be treated, on the volume of the bath, on the nature of the wood treated and on the temperature and pH of the bath, the wood is then placed in the drying facility at controlled temperature and humidity in order to carry out conventional drying which makes it possible to attain a water content of 15 to 20% for use in cooperage, starting from a wood having a water content of between 25 and 45%, preferably between 30 and 40%, before the bringing into contact with the enzymatic preparation.
The method according to the invention has proved to enable hydrolysis of soluble ellagitannins naturally present in wood, firstly ellagitannins linked to parietal polysaccharides, and also diverse aromatic molecules (such as digallic acid or heteroside coumarins) which are transformed, by the method according to the invention, into neutrally tasting molecules.
The method according to the invention therefore makes it possible to improve the impact on taste of the wood extracts by eliminating a significant portion of the undesirable compounds (such as certain phenol compounds) and hydrolysing forms judged to be bitter or astringent. This is particularly important for wood subsequently coming into contact with foods (typically, wood for cooperage).
This method superficially modifies the ultrastructure of the wood. By electron microscopy, a decrease in the cell wall is noted over 1 mm. Over 1 to 3 mm, the pores of the cells are open. The effects of the process are felt up to a depth of approximately 5 mm.
The present invention also has the advantage of providing a rapid method for ageing wood, while at the same time respecting all of the physicochemical reactions linked to the natural drying of wood.
In addition, the invention has the advantage of providing a reproducible method for seasoning wood and of allowing better homogeneity of the quality of treated wood between different species of wood or species of wood of different origin, or between the various portions of the same tree. More constant physicochemical (in particular mechanical and aromatic) properties of the wood treated according to the invention are therefore obtained, with respect to wood dried naturally, this being independent of meteorological factors.
The method according to the invention also makes it possible, through a suitable choice of enzymes having more specific activities, to confer on the wood specific properties such as the expression of specific odours or flavours.
Materials and Methods
1. Origin of the Wood Samples
The oak samples consist of the heart-wood transformed into duramen, of trees from 110 to 150 years old, derived from homogeneous and suitably maintained areas of forest. Only the lower quarter of the trunk, generally constituting the wood for cooperage, is used for the study. The samples treated with the enzymatic composition according to the invention are simply dried in a drying facility (1 month, 40xc2x0 C., with ventilation). The various randomly-taken samples are planed down to sawdust by crushing in liquid nitrogen, before being sieved so as to keep only the particles less than 250 xcexcm in size. The samples are conserved after lyophilization, to be analysed within a period of 2 months. The species studied is Quercus petraea (Allier, France).
2. Preparation of Extracts and Control of Their Composition
1 g of sawdust (250 xcexcm) is extracted with 100 ml of solvent (7:3 by volume acetone/water) for 12 h at room temperature on a shaking platform; the resulting extract is then filtered through a membrane, lyophilized and weighed.
100 xcexcg of lyophilized extract are used to identify the major ellagitannins, with an LSIMS mass spectrometer.
1 mg of the extract is taken up with methanol/water (6:4) in order, to be analysed by HPLC coupled to a UV-detector and to an LSMIS mass spectrometer, according to the device developed by Vivas et al. (1995). The HPLC separation method is described in the following paragraph.
3. Assaying of Phenol Compounds
3.1. Coumarins
The coumarins are quantitatively extracted, by extraction with diethyl ether, from 20 ml of a wood extract. The organic phase is evaporated to dryness and the residue is taken up with methanol. The HPLC analysis is carried out using a Varian 5060 coupled with a spectrofluorometric detector (Kontron SFM23/B) and an Ultrasphere ODS column. The pure coumarins were supplied by Extrasynthxc3xa8se (esculin, esculetin, scopoletin, ombelliferone, methylombelliferone) and Sigma (sporalen). The coumarins are observed by fluorescence (excitation 425 nm and emission 325 nm).
3.2. Ellagitannins
3.2.1. Reference Products
Vescalagin and castalagin are isolated and purified from the duramen of Q. robur, under the conditions described by Vivas et al. (1995). The various roburins (A-E) and grandinin originate from Scalbert (INA/INRA Thierval-Grignon).
3.2.2. Separation and Assaying of Ellagitannins by HPLC
The chromatographic technique for separating and assaying ellagitannins is in accordance with the method developed by Scalbert et al. (1990). The wood extracts are analysed by HPLC on an Ultrasphere ODS column. The detection is carried out at xcex=280 nm.
3.2.3. Assaying of Total Ellagitannins
a) Estimation of the Level of Total Phenol Compounds
The richness of the wood extracts in total phenol compounds is estimated either by the method using the Folin-Ciocalteu reagent, or by measuring the absorbance at 280 nm of the extracts diluted 100-fold (Vivas et al., 1993). These two methods give results which are comparable but not specific for ellagitannins.
b) Reaction for Oxidation with Nitrous Acid
In this method proposed by Bate-Smith (1972), esters of hexahydroxyphenic acid and of glucose are oxidized with nitrous acid under a nitrogen stream. The reaction produces a blue coloration which is measured at 600 nm. The results are estimated in mg/g or mg/l of castalagin equivalent (xcex5600 nm: 983 gxe2x88x921).
c) Acid Degradation
The method proposed is adapted from that developed by Peng et al. (1991). It is based on the acid hydrolysis of ellagitannins, followed by assaying, by HPLC, of the released ellagic acid. The results are expressed in mg/g of castalagin equivalent, taking one mole of castalagin to give, under these conditions, one mole of ellagic acid (Peng et al., 1991).
4. Extraction and Assaying of Polysaccharides
4.1. Study of the Polysaccharide Fraction of the Control and Treated Samples
4.1.1. Extraction of Polysaccharides
139 g of dry sawdust are left to soak for 72 h on a shaking platform at room temperature. The solution is then filtered and centrifuged. The extract is then concentrated down to 500 ml.
4.1.2 Isolation of Polysaccharides
The extracts undergo 3 precipitations (one at 1:9 of water/ethanol at 95% vol., two at 1:5 of the same mixture). The precipitation is carried out at 3xc2x0 C. for 12 h. Next, the fractions are lyophilized.
4.1.3. Partial Characterization
The precipitate has a frothy appearance, very pale in colour, slightly grey, which is probably a complex with the ellagitannins from which it is difficult to isolate the polysaccharide fraction.
4.1.4. Assaying Using a Chemical Method
a) Assaying of Neutral Polysaccharides (nP)
The neutral polysaccharides are assayed using the sulphuric phenol method. The optical density is read at 490 nm and the results are expressed in mg/l of glucose equivalent.
b) Assaying of Acid Polysaccharides (aP)
The acid polysaccharides are assayed using the meta-phenylphenol method. The optical density is measured at 520 nm and the results are expressed in mg/l of galacturonic acid equivalent.
4.2. Extraction and Assaying of Polysaccharides from Wood
1 g of wood shavings derived from oak trees from various regions, the wood of which has a characteristic grain, is left to soak for 24 h in water at 20xc2x0 C. on a shaking platform. The solution is collected by filtration and centrifuged. The sawdust recovered is then dried and left to soak once again, in a 5% sodium hydroxide solution, under the same conditions. The alkaline solution is also collected by filtration and centrifuged. Then, the two categories of extracts (water and sodium hydroxide) are supplemented with 95% ethanol in a proportion of 1:5 by volume. The polysaccharides are then collected by centrifugation and taken up with distilled water at 60xc2x0 C. The nP are assayed with sulphuric phenol and the aP with meta-phenylphenol. The reference solutions are, respectively, an aqueous solution at 100 mg/l of glucose and 50 mg/l of galacturonic acid for nP and aP.